Interorganelle interactions are key processes controlling eukaryotic cell function, and dysregulation of these interactions has been implicated in many human diseases. However, relatively little is known about macromolecular complexes that mediate organelle interactions, due to obstacles that have been difficult to overcome. First, many relevant proteins are integral membrane proteins, which are hard to purify and maintain weak but physiologically important binding interactions. Capturing such interactions by conventional biochemical and genetic approaches is technically difficult. Second, simultaneously tracking transient organelle populations and interactions requires the ability to follow real time dynamics in living cells using multi-color fluorescent probes. However, many fluorescent proteins (FPs) used for live imaging are compromised by the oxidizing environment of many organelles, including ER, Golgi, and secretory vesicles. The goal of this proposal is to define protein complexes that define and modulate novel organelle subpopulations, using a combination of new technologies in mass spectrometry and fluorescent protein based probes for live cell imaging. Our Specific Aims are: (1) Identify candidate protein markers of novel organelles and interorganellar protein complexes. We will develop a proteomics strategy to profile proteins within organelle subpopulations that are dynamic and transient, as well as macromolecular complexes that bridge organelles. (2) Develop novel biosensors to track these protein markers in living cells, by time resolved imaging and high resolution microscopy. We will maximize the available colors of the fluorescent protein spectrum for use in multi-color live cell imaging studies, by solving key problems in fluorescent protein reporters caused by organellar environments that restrict their folding and function. (3) Apply these methods to cutting edge problems in cell biology, addressing mechanisms underlying (i) ER stress and Ca2+-mediated organelle remodeling, (ii) Zn2+ homeostasis, and (iii) cell polarity. We will combine technologies developed in Aims 1 and 2 to create a new experimental workflow which integrates mass spectrometry/proteomics, biosensor design, and high resolution fluorescence microscopy, and apply this to relevant problems in collaborator labs in Aim 3. Our proposal establishes a unique, multidisciplinary collaboration between a team of four investigators, who are leading experts in technologies of proteomics/mass spectrometry, protein engineering and biosensor design, and cutting edge methods for high resolution cell imaging. The combined expertise from these investigators gives us a unique opportunity to discover novel organelles and macromolecular complexes involved in interorganelle contacts, and define their cell biology.